A cultured rat mast cell line, the RBL-2H3 cell, was shown to produce the cytokine, TNFalpha, at physiologically active concentrations, in response to stimulation with antigen, or the combination of Ca2+-ionophore and phorbol myristate and in cells transfected with the gene for the muscarinic m1 receptor by carbachol. Production and release of TNF` were two distinct processes although both were dependent on continued stimulation of the cell. Production of TNFalpha was stimulated by an elevation in cytosolic Ca2+ or by the activation of protein kinase C. A strong synergizing signal, possibly the activation of a tyrosine kinase, was apparent in antigen-stimulated cells. All other stimulants were weak promoters of TNFalpha production compared to antigen although all stimulants were capable of producing maximal release of secretory granules. TNF was not constitutively expressed nor incorporated into secretory granules. It was released by a process analogous to constitutive secretion in that brefeldin A, an agent known to disrupt Golgi membranes in these cells, inhibited this release without inhibiting release of secretory granules. Unlike constitutive secretion, however, the secretion of TNF was highly regulated by Ca2+ and protein kinase C. Studies with various stimulants and inhibitors indicated that simultaneous mobilization of Ca2+ and activation of protein kinase C were sufficient signals for secretion unlike production of TNF which, as noted above, was dependent on additional synergistic signals. Because suppression of Ca2+ mobilization or inhibition of protein kinase C alone abrogated TNF secretion, the process may be amenable to therapeutic intervention.